Subcellular molecular localization analysis by in situ derivatization

2018 Fiscal Year Final Research Report

• Project/Area Number: 16K21276
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: Physical pharmacy; Analytical chemistry
• Research Institution: University of Shizuoka
• Principal Investigator: Mizuno Hajime 静岡県立大学, 薬学部, 講師 (30457288)
• Research Collaborator: Todoroki Kenichiro ; Toyo'oka Toshimasa ; Ueda Kazuki ; Kobayashi Yuta ; Miyazaki Yasuto ; Shindo Takuya ; Minami Teppei ; Tanaka Natsumi ; Kato Yoshihiro
• Project Period (FY): 2016-04-01 – 2019-03-31
• Keywords: 細胞内小器官 / メタボロミクス / 誘導体化 / 質量分析

Outline of Final Research Achievements

Metabolomics is a rapidly developing field in the post-genomic research. However, the conventional metabolic approaches are difficult to know the localization of cellular metabolites because these methods are analyzing homogenized large amounts of cells or tissues. In this study, in order to analyze metabolites located in the mitochondria, we developed an analytical method for metabolites in mitochondria by using mitochondrial localized photoaffinity probe. The probe we synthesized has a diazirin as photoaffinity probe and rhodamine as a mitochondrial affinity. From the result of a high resolution laser confocal microscope observation, it was confirmed that this probe was localized in mitochondria of HepG2 cells. The cells which this synthesized probe have been treated were irradiated with UVA. As the result of LC-MS/MS analysis of intracellular metabolites after photo reaction, some metabolite peaks reacted to the photo affinity probe were detected.

Free Research Field

分析化学

Academic Significance and Societal Importance of the Research Achievements

本研究は、ミトコンドリアなどの細胞内に存在する様々な微小領域ごとに存在する代謝物という生体機能の発現と密接にかかわる分子を見つけることを目的としている。本研究で開発した方法により、これまでの方法では不可能だった代謝物の正確な細胞内局在を調べることが可能となった。これにより、これまで分からなかったミトコンドリアをはじめとした様々な細胞内小器官の詳細な機能解明が可能となり、疾患メカニズムや治療法開発につなげることができる。